Pulmonary hypertension and its associated altered vascular reactivity, are a major source of morbidity and mortality for children with congenital heart disease (CHD) and increased pulmonary blood flow. Pulmonary vascular tone is regulated, in part, by a complex interaction of vasoactive substances produced locally by the vascular endothelium, such as nitric oxide (NO) and endothelin-1 (ET-1). Endothelial injury secondary to CHD with increased pulmonary blood flow disrupts these regulatory mechanisms, and has been implicated as a potential factor in the development of pulmonary hypertension. To investigate the role of early endothelial dysfunction in the pathophysiology of pulmonary hypertension secondary to CHD, we established a unique model of pulmonary hypertension with increased pulmonary blood flow, utilizing in utero placement of a large aortopulmonary shunt in the fetal lamb. Based on our preliminary data, we hypothesize that increased pulmonary blood flow produces a progressive increase in ET-mediated vasoconstriction and mitogenesis that is initially opposed by an activated NO cascade. However, with further endothelial dysfunction secondary to increased flow and pressure, the NO cascade is decreased. This leads to unopposed ET-mediated vasoconstriction and mitogenesis and the progressive development of pulmonary hypertension and vascular remodeling. In this application, we will utilize our unique animal model, and a sequential and integrated physiologic, biochemical, molecular, and anatomic approach, to investigate this hypothesis. Specifically, we will examine: (a) the normal maturational changes in the NO and ET-1 cascades during the first two months of life; (b) the role of early alterations in the NO and ET-1 cascades in the development of pulmonary hypertension secondary to increased pulmonary blood flow; and (c) the interactions between the NO and ET-1 cascades, and their regulatory mechanisms, in the normal and abnormal pulmonary vasculature. Correlations between endothelial dysfunction, increased vascular reactivity, and vascular remodeling will be made, and predictors of increased vascular reactivity will be sought. This information may improve our ability to recognize early pulmonary vascular changes, and better predict those patients with CHD who will suffer increased peri- and post-operative morbidity secondary to increased vascular reactivity. This would profoundly affect the timing and feasibility of surgical correction, and should lead to improved treatments and prevention strategies for pulmonary hypertension.